1. Field of The Invention
The present invention is directed to a method for the renaturation, reassociation or hybridization of single stranded nucleic acid molecules into double stranded nucleic acid molecules wherein the rate of reaction is greatly increased over the rate of reaction under standard reference conditions of 0.12M phosphate buffer at 60.degree. C. More particularly, the present invention is directed to a method for the renaturation, reassociation or hybridization of nucleic acids, including DNA to DNA, RNA to DNA, and RNA to RNA reactions wherein the rate of the reaction is greatly increased by factors of 50 to 100 times, and even up to several thousand times that of the reaction rates observed under standard reference conditions. These greatly accelerated reaction rates are achieved through the utilization of reaction solutions containing nucleic acid precipitating agents.
2. Description of The Prior Art
Numerous methods for the nucleation of single stranded nucleic acid molecules into double stranded molecules are known in the art and have proven to be useful tools for the analysis of genetic material from a wide variety of organisms. Generally speaking, these nucleation reactions, renaturation, reassociation and hybridization, are based on the tendency of single stranded nucleic acid molecules having blocks or segments of complementary base sequences to form base paired structures between these complementary sequences and to rewind forming double helices. The greater the extent of sequence complementarity between the single stranded nucleic acid molecules, the greater the tendency for a given pair of molecules to nucleate and form a double stranded or duplex molecule.
Renaturation, reassociation and hybridization are essentially synonymous reactions between single stranded nucleic acid molecules. As such, they will be discussed interchangeably throughout the body of this paper. However, the following distinction may prove helpful in understanding the technology involved. Renaturation generally refers to the formation of a double stranded nucleic acid molecule from two single stranded molecules which were originally base paired to one another and separated through a denaturation process. Reassociation refers to the process of forming double stranded nucleic acid molecules between two single stranded molecules which usually come from different original molecules. Hybridization refers to the formation of double stranded nucleic acid molecules from single stranded nucleic acid molecules of individual origin with respect to one another. It should be appreciated that these are not clear cut distinctions and that there is considerable overlap between them. For example, DNA:DNA reactions are commonly called both reassociation and hybridization reactions. On the other hand, the formation of an RNA:RNA double stranded molecule is generally referred to as hybridization.
The kinetics of these reactions are well understood in the art also following second-order kinetics. Thus, as the concentration of the single stranded nucleic acid molecules is increased, the rate of the reaction is also increased. Conversely, decreasing the concentration of the single stranded nucleic acid reactant will decrease the rate of reaction and thus increase the time necessary for the formation of the double stranded nucleic acid molecules to take place.
The effect of temperature on the reaction rate is also well known in the art. As the temperature of the reaction decreases below the T.sub.m (the temperature at which 50% of the double stranded molecules is denatured, also known as the "melting temperature") a maximum rate for the reaction is achieved at temperatures of approximately 15.degree. C. to 30.degree. C. below the T.sub.m. Further decreases in temperature are known to decrease the rate below this maximum rate.
Lastly, with respect to the kinetics of these reactions, it is known that the reaction rates are very dependent on the ionic strength below 0.4M for electrolytes such as NaCl and are almost independent of the salt concentration above this ionic strength.
More information on the kinetics and reaction rates of these nucleic acid association reactions can be found in the following publications:
Wetmur, R., and Davidson, N. (1968), J. Molec. Biol. 31, 349;
Wetmur, R. (1975), Biopolymers 14, 2517;
Britten, R., J., Graham, D., and Neufeld, B. (1974), Methods Enzymol, 29, 363;
Kohne, D. E., Levinson, S. A., and Byers, M. J. (1977) Biochemistry 16, 5329; and
Orosz, J. M., and Wetmur, J. G., (1977) Biopolymer 16, 1183.
It has long been recognized in the art that a major limitation on the utility of these known nucleic acid association techniques is the basic rate of the reaction. Reaction times on the order of several hours to tens of hours and even days are commonplace. Increasing the reaction rate by increasing the quantities of single stranded nucleic acid molecules utilized in the reactions (due to the second-order kinetics) is not a desirable solution to this problem for three reasons. First, in many cases the target single stranded nucleic acid in the reaction is extracted from physiological samples which inherently limits the amount of such nucleic acid available to that contained in the cells of the physiological sample. Secondly, there are significant expenses associated with the use of nucleic acid reactants which limits the practical utility of increasing the quality of reactants. Thirdly, increasing the quantities of single stranded nucleic acid molecules decreases the sensitivity of the reaction by increasing the background noise. Nonetheless, a number of techniques have been developed to increase the basic rate of these reactions by factors of 5 to 50 or more. Techniques of limited applicability have also been developed which increase the basic reaction rate by factors on the order of 1000 or more. However, as will be discussed in detail below, none of these prior art techniques has been successful at producing greatly accelerated reaction rates of 50 to 100 times or more than the basic reference standard reaction in a single phase system applicable to DNA:DNA, DNA:RNA, and RNA:RNA reactions.
When dealing with reaction rates, the accepted standard reference condition for the comparison of these rates is an aqueous solution of 0.12M phosphate buffer (PB) at 60.degree. C. A similar standard reference condition that is often used giving comparable reaction rates is an aqueous solution of 0.18M NaCl at 60.degree. C.
By far the most common technique of accelerating the reaction rate above that of the standard reference condition has been to increase the salt concentration of the reaction solution above that of the standard reference condition. As detailed in following table, while significant reaction rate increases are observed by increasing the salt concentration, the prior art techniques indicate that the rate of increase levels off or even falls for salt concentrations above 2M.
TABLE 1 ______________________________________ PRIOR ART KNOWLEDGE OF EFFECT OF SALT CONCENTRATION ON DNA:DNA HYBRIDIZATION RATES Rate increase relative to 0.18M Salt NaCl reference condition ______________________________________ A. Sodium Chloride 0.18M 1 0.72M 5.8 1M 7 1.2M 7.7 Britten, et al. 1.85M 8.6 3.2M 12.3 4.75M 21 B. Cesium Chloride 1M 7.6 4M 12.7 7.5M 15.6 C. Sodium Phosphate 0.12 (0.18M Na) 1 0.48M (0.72M Na) 5.6 Britten, et al. and 1M (1.5M Na) 8.4 Wetmur and Davidson 1.23M (1.85M Na) 10.1 2.1 (3.2M Na) 12.1 D. Sodium Perchlorate 1M 11 2.2M 6.8 Wetmur and Davidson 4.0M 3.4 5.2M 1.5 6.4M 0.7 E. Lithium Chloride 0.4M 3.9 Orosz and Wetmur 1M 11.6 F. Potassium Chloride 0.7M 5.3 1M 5.8 2M 5.4 Orosz and Wetmur 3M 10.0 4M 11 G. Sodium Bromide 3M 9 Orosz and Wetmur H. Sodium Sulfate 3M 9 Orosz and Wetmur I. Ammonium Chloride 4M 30 ______________________________________
While the data in Table 1 relates to the rate increases found with respect to DNA:DNA reactions, it will be appreciated that the reaction rates of RNA with DNA are reported as being less affected by changes in salt concentration. Other researchers have demonstrated that for salt concentrations above the standard reference conditions, the relative rate of RNA:DNA reaction is affected to about one-half the extent of those rates found for DNA:DNA reactions when the RNA used has comparatively little secondary structure. For RNA reactants with more secondary structure, the effect of elevated salt concentration has been found to be even less. In fact, no change in rate is observed for hybridization of excess RNA with DNA over comparative ranges of salt concentrations (see, e.g., Van Ness, J. and Hahn, W. E. (1982) Nucl. Acids. Res. 10, 8061). While little data is available for RNA:DNA hybridization where the DNA is the excess reactant, it is commonly assumed in the art that the effect of elevated salt concentration on such a reaction system is comparable to that of the excess RNA system.
An alternative approach to the acceleration of the rate of these nucleic acid association reactions is the previously developed two-phase phenol aqueous emulsion technique for the reassociation of DNA to DNA (Kohne, D. E., Levinson, S. A., and Byers, M. J. (1977) Biochemistry 16, 5329). In this two-phase system, the agitation of an emulsion formed between phenol and an aqueous salt solution has produced greatly accelerated reaction rates over 100 times faster than comparative standard condition rates. However, the two-phase phenol emulsion technique has not produced similarly greatly accelerated reaction rates for RNA:RNA and RNA:DNA systems. The greatest reaction rate increase observed for RNA:RNA and RNA:DNA reactions is only 50 to 100 times that of the standard reference condition rate. This technique is further limited in that reaction will not occur unless an emulsion is present and agitated and the reaction temperature is below 75.degree. C.
A number of other techniques for producing reaction rate increases on the order of 10 fold above the standard reference condition rate have utilized the volume exclusion principle to promote the acceleration of the reaction rate. These techniques utilize the synthetic polymers polyethylene glycol, dextran, or dextran sulfate to reduce the volume of reaction solution available to the nucleic acid reactants and thereby increase their effective concentration. However, while reaction rate increases of 10 to 15 fold over the standard reference condition rate for DNA:DNA reactions have been reported, rate increases of only about 3 fold are reported for RNA:DNA reactions. Details of these techniques can be found in the following publications:
Renz, M., and Kurz, C. (1984) Nucl. Acids Res. 12, 3435; and
Wahl, G. M., Stern, M., and Stark, G. R. (1979) Proc. Natl. Acad. Sci. USA 75, 3683.
Accordingly, it is a principal object of the present invention to provide a method for the renaturation, reassociation, or hybridization of nucleic acids that produces a greatly accelerated reaction rate on the order of 100 or more times that of the standard reference condition rate and that is applicable to DNA:DNA, RNA:DNA, or RNA:RNA reaction systems. Additionally, it is a further object of the present invention to provide a method that promotes greatly accelerated reaction rates without requiring the utilization of a two-phase system or the formation of an emulsion. It is a further object of the present invention to provide a method wherein greatly accelerated reaction rates are obtainable without the need to increase the concentrations of single stranded nucleic acid reactants. Lastly, it is an additional object of the present invention to provide a method for greatly accelerating the rate of these nucleic acid association reactions that is widely applicable to a variety of reaction mixture volumes and hybridization temperatures.